This invention relates to methods and compositions for modulating adhesion between cells that express E-cadherin (e.g., epithelial cells, endothelial cells) and T lymphocytes (e.g., intraepithelial T lymphocytes). In particular, the invention relates to methods and compositions for modulating binding between E-cadherin and heterotypic cognates of E-cadherin, such as xcex1Excex27 integrin.
The adhesive interactions of cells with other cells and between cells and the extracellular matrix are believed to play critical roles in a wide variety of processes including, for example, modulation of the immune system, regulation of developmental processes and tumor progression and metastasis. These interactions are mediated by adhesion molecules which transduce information from the extracellular to the intracellular matrix.
Three families of adhesion molecules which mediate these interactions have been identified: the integrins, the cadherins and the selections. In general, adhesion molecules are transmembrane proteins which contain an extracellular domain for interacting with an extracellular matrix or cellular component, a transmembrane domain spanning the cell membrane and a cytoplasmic domain for interacting with one or more cytoskeletal components.
The integrins represent one of the best characterized families of adhesion receptors. Integrins are glycoprotein heterodimers which contain a noncovalently-associated xcex1 and xcex2 subunit. There are fourteen known xcex1 subunits and eight known xcex2 subunits which can pair to form at least twenty different integrin molecules. Several distinct integrin xcex1 chains are capable of pairing with one type of xcex2 chain to form a xcex2 chain subfamily.
Recently, Parker et al. described a novel integrin heterodimer that is expressed on intra-epithelial T lymphocytes (iIEL), i.e., the population of T lymphocytes located along the basolateral surfaces of the epithelial cells which line the mucosa, adjacent to the epithelial cell basement membrane. (Parker, C. et al. (1992) Proc. Natl. Acad. Sci. USA 89:1924). Originally defined by an antibody which recognizes the human mucosal lymphocyte 1 antigen (HML-1), the novel integrin is present on  greater than 90% of intestinal IEL (iIEL) and on approximately 40% of lamina propria T lymphocytes (which lie between the epithelial basement membrane and the muscularis mucosae) (Cerf-Bensussan, N. et al., (1987) Eur. J. Immunol. 17, 1279-1285). The HML-1 antigen contains a novel xcex1 chain (designated xcex1E, for xe2x80x9cepithelial associatedxe2x80x9d) associated with a xcex27 chain (Parker, C. et al. (1992) Proc. Natl. Acad. Sci. USA 89:1924). Although the xcex1E and xcex27 subunits have been cloned, a receptor for the xcex1Excex27 integrin has not been identified. (See U.S. patent application Ser. No. 08/199,776, the contents of which are incorporated herein by reference, which discloses the primary structure of the xcex1E chain and Yuan Q. et al. (1990) Int. Immunol. 2:1097; Erle, D. J. et al., (1991) J. Biol. Chem. 266, 11009-11016, which disclose a HML-1 xcex27 chain clone).
The cadherins play an important role in the establishment and maintenance of intercellular connections between cells of the same type (reviewed in Geiger B. et al. (1992) Annual Review of Cell Biology 8:307; Kemler R. (1993) Trends in Gastroenterology 9:317; Takeichi M. (1990) Annual Review of Biochem. 59:237; Takeichi M. (1991) Science 251:1451). The cadherins are synthesized as precursors that are cleaved during post-translational processing. The mature cadherins are single chain molecules which include a relatively large extracellular domain (typically divided into five sections or xe2x80x9cectodomainsxe2x80x9d), a single transmembrane region and a cytoplasmic tail. Sequence analysis of cadherin cDNA clones reveals that the extracellular cadherin amino acid sequence can be divided into three inter-homologous ectodomains (xe2x80x9cEC 1-3xe2x80x9d), each of which is 110 amino acids in length (Takeichi M. (1990) Annual Review of Biochem. 59:237). Within the ectodomains, characteristic sequences of four to five amino acids (LDRE and DXNDN) are well conserved among all cadherins. In particular, the sequence DXNDNXP, present in EC 1-3, is thought to bind divalent calcium and is generally believed to be essential for cadherin function. Two additional, less well conserved domains (xe2x80x9cEC 4-5xe2x80x9d) are located proximal to the membrane. Among the classical cadherins (i.e., P-(placenta), E-(epithelial), and N-(neural) cadherin), the cytoplasmic domain contains the highest degree of homology, followed by the EC-1 domain (Takeichi M. (1990) Annual Review of Biochem. 59:237). The high degree of homology observed for the cytoplasmic domain reportedly is a reflection of the association of cadherins with a group of intracellular proteins, xe2x80x9cthe cateninsxe2x80x9d, that stabilize cadherin active conformation (Kemler R. (1993) Trends in Gastroenterology 9:317).
It is generally believed that sequences in the EC-1 extracellular domain are necessary to mediate homotypic (i.e., cadherin-to-cadherin) binding. Swapping experiments in which part of the E-cadherin molecule is replaced with a corresponding portion of the P-cadherin molecule have been used to identify the amino acid portions of post-translationally processed cadherin that are required for biological activity. (Nose A. et al. (1990) Cell 61:147). In particular, Nose et al. report that an HAV tripeptide sequence is essential for homotypic cadherin binding. Further, Takeichi report that the amino acid residues flanking the HAV tripeptide sequence also contribute to homotypic binding specificity. (Takeichi M. (1991) Science 251:1451). A review of the literature indicates that research directed to understanding cadherin-mediated adhesion has focussed on efforts to elucidate the mechanism underlying cadherin-mediated homotypic cell adhesion. Little attention has been directed to understanding what, if any, role is played by cadherin in heterotypic cell-to-cell adhesion.
While it has been known for some time that integrins and other adhesion molecules function in immune system modulation, e.g., by playing a role in the adhesion of peripheral lymphocytes to endothelium and in homing to lymph nodes, relatively little is known regarding the mechanism by which lymphocytes home and transmigrate through the vascular endothelium to specifically target tissue locations. In particular, little is known about the molecules that function in mucosal T lymphocyte homing and adhesion, the subset of the general immune system which includes the lymphocytes which populate the gastrointestinal, genito-urinary and respiratory tracts, and the mammary glands. (see, Cepek, K. et al., (1993) J. Immunol. 150, 3459-3470 and references cited therein). In part, the difficulties encountered in cloning the xcex1E subunit (described in U.S. patent application Ser. No. 08/199,776) and in obtaining anti-epithelial cell antibodies which block intra-epithelial lymphocyte adhesion have hindered the identification of an epithelial cell receptor for the intra-epithelial lymphocyte xcex1Excex27 integrin. An incomplete understanding of the role played by cytokines in modulating xcex1Excex27 expression in intra-epithelial cells, also has impeded the identification of a receptor for the xcex1Excex27 integrin.
Compositions and methods for modulating the interaction between a T lymphocyte and an E-cadherin expressing cell are disclosed herein. The interaction is dependent upon the specific binding of E-cadherin to a heterotypic cognate of E-cadherin that is present on the T lymphocyte. Although the homotypic adhesion of one cadherin molecule for another cadherin molecule is well known in the art, the phenomenon of heterotypic cadherin interaction, i.e., the specific binding between cadherin and a non-cadherin molecule, is new.
In view of our observations: (1) that E-cadherin functions as a receptor for xcex1Excex27 integrin, an exemplary adhesion molecule that is preferentially expressed in intra-epithelial T lymphocytes (xe2x80x9cIELxe2x80x9d) and (2) that E-cadherin is expressed on the vascular endothelium, we believe that E-cadherin plays a key role in the interactions between T lymphocytes and E-cadherin expressing cells and in particular, in the interactions of intra-epithelial T cells and epithelial or endothelial cells. Accordingly, the specific examples disclosed herein (primarily directed to fully understanding the interaction between E-cadherin and xcex1Excex27) serve as a model for other E-cadherin mediated T lymphocyte adhesion reactions.
Although not intending to be bound by any particular theory, it is believed that E-cadherin mediated binding of T lymphocytes to the vascular endothelium is involved in the migration of lymphocytes into mucosal tissue. It further is believed that the interaction of xcex1Excex27 integrin with epithelial cell E-cadherin is involved in the retention of IELs within the epithelium. The dependence of lymphocyte transmigration and adhesion on tissue specific cadherin expression has not previously been reported. Accordingly, the invention provides compositions and methods for inhibiting the transmigration of T lymphocytes through the vascular endothelium into mucosal tissue, and for inhibiting lymphocyte adhesion to E-cadherin expressing epithelial cells. Thus, the invention is useful for modulating a mucosal immune response in vivo.
One aspect of the invention is directed to isolated peptides which inhibit adhesion between T lymphocytes (e.g., IELs) and E-cadherin expressing cells (e.g., epithelial cells, endothelial cells) in vivo and/or in vitro. The peptides specifically bind to xe2x80x9cheterotypic cognates of E-cadherinxe2x80x9d, i.e., non-E-cadherin ligands which specifically bind to E-cadherin. According to one embodiment, the peptides have sequences which are related to, or derived from, the amino acid sequence of the extracellular domain of E-cadherin, i.e., that portion of the E-cadherin protein which is exposed to the extracellular environment when the protein, expressed on mucosal epithelial cells, is in its native conformation. E-cadherin includes an extracellular domain (amino acids 1-552 of Sequence I.D. Nos. 1 and 2), a transmembrane domain (amino acids 553-576) and a cytoplasmic domain (amino acids 577-726). By binding to the heterotypic cognate of E-cadherin on T lymphocytes (e.g., by binding to xcex1Excex27 integrin on IEL), the E-cadherin derived peptides inhibit binding of the T lymphocyte to the E-cadherin expressing cell. The peptides can be also specifically reactive with a monoclonal antibody that binds to E-cadherin and that can inhibit T lymphocyte binding.
According to another aspect of the invention, monoclonal antibodies that inhibit adhesion between a T lymphocyte, such as an IEL and an E-cadherin expressing cell, such as an epithelial cell or an endothelial cell, are provided. The monoclonal antibodies specifically bind to a cadherin, more preferably, to E-cadherin. The antibodies are useful for blocking a functional activity of the E-cadherin, such as an in vivo functional activity (e.g., retention of the IELs in the epithelium) or an in vitro functional activity (e.g., adhesion of IELs to an epithelial cell monolayer as determined in an adhesion assay). Accordingly, the antibodies are useful as reagents in assays for screening molecular libraries to identify lead compounds that modulate adhesion between T lymphocytes and E-cadherin expressing cells.
According to yet another aspect of the invention, a pharmaceutical preparation for modulating a mucosal immune response in a subject is provided. The composition includes a pharmaceutically acceptable carrier and an agent that inhibits adhesion between a T lymphocyte and an E-cadherin expressing cell. The agent (e.g., the above-described isolated peptide, isolated E-cadherin or monoclonal antibody) is present in a therapeutically effective amount for treating the mucosal immune response. Preferably, the isolated E-cadherin is soluble E-cadherin.
According to another aspect of the invention, a method for screening a molecular library to identify lead compounds that modulate adhesion between a T lymphocyte and an E-cadherin expressing cell is provided. The method involves performing an adhesion assay (e.g., between an IEL and an epithelial cell) in the presence and absence of at least one member of the molecular library and comparing the adhesion assay results to determine whether the molecular library member modulates adhesion between the T lymphocyte and the E-cadherin expressing cell. The adhesion screening assays of the invention also embrace assays in which an isolated T lymphocyte ligand (alternatively referred to herein as xe2x80x9ca heterotypic cognate of E-cadherinxe2x80x9d) and an isolated receptor E-cadherin (described below) serve as the binding partners in the adhesion assay. Optionally, the isolated receptor E-cadherin and/or its heterotypic cognate may be immobilized onto a solid support, such as a microtiter plate.
According to another aspect of the invention, an antibody assay for screening a molecular library is provided. The assay is useful for identifying pharmaceutical lead compounds that modulate adhesion between a T lymphocyte and an E-cadherin expressing cell. The antibody assay is performed, for example, by contacting a receptor E-cadherin with an antibody (known to specifically bind to the receptor E-cadherin and inhibit the interaction between T lymphocytes and E-cadherin expressing cells) in the presence and absence of at least one member of the molecular library and determining whether the library member modulates binding between the antibody and the receptor E-cadherin. As used herein, a xe2x80x9creceptor E-cadherinxe2x80x9d refers to an E-cadherin expressing cell, an isolated E-cadherin or an isolated peptide related to, or derived from, the extracellular domain of E-cadherin that is capable of specifically recognizing and binding to a ligand (e.g., xcex1Excex27 integrin) expressed on a T lymphocyte.
According to yet another aspect of the invention, a method for inhibiting adhesion between a T lymphocyte and an E-cadherin expressing cell is provided. The method involves contacting the E-cadherin expressing cell with an agent (e.g., the above-described monoclonal antibodies, isolated peptides or isolated E-cadherin) that inhibits adhesion between E-cadherin and its heterotypic cognate.
According to another aspect of the invention, a method for modulating the mucosal immune response of a subject is provided. The method involves administering to the subject a pharmaceutical composition containing the above-described agents for inhibiting adhesion between a T lymphocyte and an E-cadherin expressing cell.
These and other aspects of the invention, as well as various advantages and utilities, will be more apparent with reference to the detailed description of the preferred embodiments.